Project Summary/Abstract Astrocytes constitute at least one third of all cells in the human brain and are critical for the development and function of the central nervous system. Reactive astrogliosis is a spectrum of cellular, molecular, and functional changes of astrocytes found in a wide range of injuries and diseases, including epilepsy, brain tumor, Alzheimer?s disease, Parkinson?s disease, stroke, inflammation, and traumatic brain injuries. Based on studies of mouse models, reactive astrocytes play both beneficial and harmful roles in disease progression and neural repair by secreting cytokines that regulate immune cells, producing growth factors, and forming scars that insulate disease tissue from healthy tissue. However, little is known about the molecular and cellular changes of astrocytes in human patients, due in part to the difficulties of purifying and culturing human astrocytes. Previous methods of purifying human astrocytes rely on serum, which induces reactive astrogliosis in the purification procedure, making it difficult to investigate reactive changes of astrocytes in patients. We recently developed a novel purification and culturing method for human astrocytes without serum. Using our new method, we will perform molecular characterization of reactive astrocytes purified from human patients with epilepsy, brain tumor, Alzheimer?s disease, Parkinson?s disease, and arteriovenous malformation. In Aim 1, we will characterize the transcriptome of reactive astrocytes and test the hypothesis that the molecular phenotypes of reactive astrocytes are diverse in humans. In preliminary studies, we found that instead of being a single state, there are diverse reactive states of astrocytes depending on the disease condition. We will examine the function of molecules induced in reactive astrocytes in humans using in vitro cultures of human astrocytes. In Aim 2, we will directly compare the responses of human and mouse astrocytes to a variety of harmful stimuli. Our preliminary data showed that human and mouse astrocytes have different susceptibility to oxidative stress and that harmful stimulus activates different signaling pathways in human vs. mouse astrocytes. These studies has the potential to reveal what reactive astrocytes do or fail to do in human neurological disorders, and provide new therapeutic targets for treating epilepsy, brain tumor, and neurodegenerative disorders.